Ultrasensitive digital immunosensing method

Abstract

An ultrasensitive digital immunosensing method, comprising: coupling magnetic particles to a primary antibody; using a single-stranded signal DNA to modify a secondary antibody; the primary antibody and the secondary antibody respectively recognizing a target protein to form a magnetic particle immune complex having a sandwich structure; separating and removing magnetic particles to obtain a free immune complex having the sandwich structure; using a droplet generator to load the free immune complex having the sandwich structure into water-in-oil (W / O) droplets; amplifying the single-stranded signal DNA to generate fluorescent droplets; and counting the fluorescent droplets to obtain the content of the target protein. The method involves simple operations, has low costs, wide applicability, high sensitivity and strong specificity, and has high value for applications such as in vitro diagnostics, screening, and biomarker discovery.

Description

An ultrasensitive digital immunosensing method Technical Field

[0001] The invention belongs to the field of analytical sensing, and mainly relates to an ultra-sensitive digital immunosensing method. Background Art

[0002] Ultrasensitive detection of proteins in complex media is crucial for medical diagnosis, biomarker discovery, and drug development. For example, in neurogenic diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD), protein biomarkers are present at extremely low levels in peripheral blood due to the presence of the blood-brain barrier. Quantitative detection of these biomarkers using traditional analytical methods such as enzyme-linked immunosorbent assay (ELISA) or chemiluminescence is challenging. Consequently, clinical diagnosis of PD and AD currently relies on medical imaging and pathological analysis rather than molecular profiling. In recent years, ultrasensitive immunosensing has made rapid progress, with representative technologies such as single-molecule immunosensing and ultrasensitive electrochemiluminescence. Among these, digital immunosensors have attracted increasing attention for their ultrahigh sensitivity (e.g., sub-femtomol detection). In principle, digital immunosensors rely on randomly partitioning a target mixture into a large number of microwells or droplets and then performing statistical analysis (e.g., Poisson distribution) of positive signals. Currently, digital immunosensors have been applied to early cancer screening, detection and discovery of low-abundance protein biomarkers, and other medical diagnostics.

[0003] Currently, a major challenge facing most digital immunosensors is the use of a microchip-based platform, which leads to two problems: the first is the high cost of manufacturing micro-nanostructure chips; the second is the use of microfluidic pumps, which makes it difficult to be widely used in clinical settings.

[0004] Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide an ultrasensitive digital immunosensing method that combines immune PCR with a droplet generation device to achieve accurate detection of low-abundance target protein molecules.

[0006] The technical solutions for achieving the purpose of the present invention are as follows:

[0007] An ultrasensitive digital immunosensing method, wherein magnetic particles are coupled to primary antibodies; the secondary antibodies are modified with single-stranded signal DNA; the primary and secondary antibodies respectively recognize the target protein to form a magnetic particle immune complex with a sandwich structure;

[0008] Separating and removing the magnetic particles to obtain free sandwich-structured immune complexes; loading the free sandwich-structured immune complexes into water-in-oil (W / O) droplets through a droplet generator;

[0009] Amplify single-stranded signal DNA to generate fluorescent droplets; count the fluorescent droplets to obtain the content of the target protein.

[0010] The magnetic particles are gold-coated magnetic nanoparticles (Au@MNPs).

[0011] The magnetic particles and primary antibodies are coupled using double-stranded DNA with different groups at both ends. Gold-sulfur covalent bonds are formed between the magnetic particles and the double-stranded DNA, and amide bonds are formed between the primary antibodies and the double-stranded DNA.

[0012] The single-stranded signal DNA modified secondary antibody utilizes the mutual covalent binding between biotin and streptavidin.

[0013] The double-stranded linked DNA is treated with a restriction endonuclease-specific enzyme digestion method to release the sandwich-structured immune complex.

[0014] The method described herein loads free immune complexes into water-in-oil (W / O) droplets via a droplet generator, wherein the droplet generator includes a centrifugal type, a microfluidic chip type, or an oscillating type.

[0015] The single-stranded signal DNA amplification includes real-time fluorescence quantitative PCR and loop-mediated isothermal amplification; the DNA amplification dye includes double-stranded DNA binding dye or molecular probe.

[0016] The target protein may be derived from animals, plants, microorganisms, or artificial products.

[0017] The target protein is sourced from untreated or pretreated human body fluids, including serum, urine, cerebrospinal fluid, or saliva.

[0018] The method described, the steps are as follows:

[0019] 1) The primary antibody is conjugated to gold-coated magnetic nanoparticles using double-stranded DNA to capture the target protein;

[0020] 2) The secondary antibody is modified with single-stranded signal DNA to recognize the target protein and form an immune complex with a gold-coated magnetic nanoparticle-sandwich structure;

[0021] 3) Use restriction endonucleases to specifically cleave the double-stranded DNA to release the sandwich-structured immune complex;

[0022] 4) using a droplet generator to load the free immune complexes into droplets, so that each droplet contains only one sandwich-structured immune complex;

[0023] 5) Performing digital PCR amplification on the droplets to generate fluorescent signals;

[0024] 6) Count the fluorescent droplets and total droplets to obtain the concentration or content of the target protein.

[0025] Beneficial effects of the present invention:

[0026] The present invention applies immune PCR to a microdroplet generating device, thereby realizing protein immunoassay at the aM protein molecule level or even the single molecule level.

[0027] The immunosensing method reduces the nonspecific adsorption caused by solid phase carriers such as Au@MNPs; digital immunosensing can only proceed normally when the connection and signal parts are present at the same time, further improving the specificity of the immunosensing.

[0028] The immunosensing method described above cleaves the double-stranded nucleic acid at specific sites to separate the gold-coated magnetic nanoparticles and the immune complex. This part of the double-stranded nucleic acid can be replaced with materials such as controllable photochemical response to release the immune complex faster and more efficiently.

[0029] The immunosensing method can use the polymerase chain reaction method to generate detection signals, and the diversity of nucleic acids themselves provides more possibilities for the joint detection of proteins.

[0030] This immunosensing method has the advantages of simple operation, low cost, wide applicability, high sensitivity and strong specificity. It can be applied to multiple fields and multiple species, and is especially valuable for in vitro diagnosis, screening, biomarker discovery and other applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG1 is a flow chart of an ultrasensitive digital immunosensing method of the present invention.

[0032] FIG2 is a representation of the nanoparticles used in the present invention during preparation.

[0033] FIG3 is a diagram showing the detection results of α-synuclein in a buffer solution according to the present invention.

[0034] FIG4 is a graph showing the detection results of α-synuclein in serum according to the present invention.

[0035] FIG5 is a diagram showing the specific detection of the present invention.

[0036] FIG6 shows the detection of phosphorylated α-synuclein ser129 according to the present invention. DETAILED DESCRIPTION

[0037] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described below with reference to the accompanying drawings.

[0038] As shown in FIG1 , an ultrasensitive digital immunosensing method comprises the following steps:

[0039] (1) Formation of immune complexes

[0040] Magnetic particles are coupled to primary antibodies;

[0041] The secondary antibody is modified with single-stranded signal DNA;

[0042] The primary antibody and secondary antibody recognize the target protein to form a magnetic particle immune complex with a sandwich structure;

[0043] Separating and removing the magnetic particles to obtain free sandwich-structured immune complexes;

[0044] (2) Droplet generation

[0045] The free sandwich-structured immune complexes were loaded into water-in-oil (W / O) droplets using a droplet generator;

[0046] Amplify single-stranded signal DNA to generate fluorescent droplets;

[0047] (3) Single molecule counting

[0048] The fluorescent droplets are counted to determine the content of the target protein.

[0049] Preferably, the magnetic particles are gold-coated magnetic nanoparticles (Au@MNPs).

[0050] Preferably, the magnetic particles and the primary antibody are coupled using double-stranded DNA with different groups at both ends, a gold-sulfur covalent bond is formed between the magnetic particles and the double-stranded DNA, and an amide bond is formed between the primary antibody and the double-stranded DNA.

[0051] Preferably, the single-stranded signal DNA modified secondary antibody uses the mutual covalent binding between biotin and streptavidin.

[0052] Preferably, the double-stranded linked DNA is treated with a restriction endonuclease-specific digestion method to release the sandwich-structured immune complex.

[0053] Preferably, the method described herein loads free immune complexes into water-in-oil (W / O) droplets via a droplet generator, wherein the droplet generator types include centrifugal type (such as CN216688147U, etc.), microfluidic chip type (such as CN113275048A, etc.) or oscillating type (such as CN108654706A, etc.).

[0054] Preferably, the method for amplifying the single-stranded signal DNA includes real-time fluorescence quantitative PCR and loop-mediated isothermal amplification.

[0055] Preferably, in the step of amplifying single-stranded signal DNA, the DNA amplification dye includes a double-stranded DNA binding dye (such as SYBR Green, ROX) or a molecular probe (such as Tapman).

[0056] The source of the target protein includes untreated or pretreated human body fluids, and the body fluids include serum, urine, cerebrospinal fluid, or saliva.

[0057] Example 1

[0058] Preparation of magnetic core particles: 360 mg of FeCl₃·6H₂O (1.33 mM) and 131.94 mg of MnCl₂ were added to 20 mL of a 1:1 mixture of diethylene glycol (DEG) and ethylene glycol (EG). The mixture was stirred for 20 minutes until the solution became transparent. 2 g of polyvinylpyrrolidone (PVP) powder was added, and the mixture was heated at 120°C until the PVP powder was completely dissolved. Subsequently, 1.5 g of sodium acetate was added and stirred for 30 minutes. The solution was transferred to a reactor and reacted at 205°C for 10 hours. After the reaction was complete, the synthesized MnFe₂O₄ magnetic nanoparticles were washed five times with ethanol and dried under vacuum for 24 hours to obtain a brown powder.

[0059] Preparation of gold seed nanoparticles (~15 nm): Heat a conical flask containing 200 mL of deionized water, add 11.11 mL of sodium citrate solution (5 mM), and bring the solution to a boil at 1200 rpm. Add 2.22 mL of chloroauric acid solution (25 mM) and stir for 5 minutes. Subsequently, add 5 mL of freshly prepared NaBH4 (100 mM) and continue stirring at 25°C for 4 hours. Store the resulting product at 4°C until ready for use.

[0060] Formation of gold-coated magnetic nanoparticles (Au@MNPs): Gold seed particles were adsorbed onto the surface of the magnetic core, and a gold shell was grown on the surface of the magnetic core using a gold seed growth method. 100 mg of MnFe2O4 magnetic nanoparticles were mixed with 200 mL of polyetherimide (PEI) solution (5 mg / mL) and sonicated for 20 minutes. 200 mL of Au seed solution was added to the mixed solution and sonicated for 30 minutes. The nanoparticles were then magnetically separated and washed five times with water. The product was dispersed in 100 mL of water to obtain 1 mg / mL Auseeds-PEI@MnFe2O4. 250 mg of PVP was added to 5 mL (0.25 mg / mL) of Auseeds-PEI@MnFe2O4 solution and completely dissolved. 200 mL of hydroxylamine hydrochloride solution was then added, and the mixture was sonicated for 3 minutes. An appropriate amount (~500 μL) of 25 mM chloroauric acid solution was then added and reacted for 5 minutes to form Au@MnFe2O4. The cells were separated by magnetic separation and washed three times with deionized water and stored at 4 °C.

[0061] The morphology, size, and zeta potential of the synthesized nanoparticles were characterized by scanning electron microscopy (SEM, Carl Zeiss AG, GeminiSEM 300) and dynamic light scattering (DLS, Malvern Panalytical, Zetasizer Nano ZS). The absorption peaks of the nanoparticles were measured by UV-Vis spectrometry (Shimadzu Corporation, UV-2550).

[0062] Characterization of nanoparticles: As shown in parts (1)-(2) of Figure 2, the synthesized gold-coated magnetic nanoparticles are spherical, with a diameter of ∼130 nm before Au coating, which increases to ∼160 nm after the gold shell is formed. Dynamic light scattering measurements show that the average size of Au@MNPs is approximately 700 nm (part (3) of Figure 2), which may be due to the aggregation of nanoparticles. The UV-visible absorption spectrum of the nanoparticles is shown in part (4) of Figure 2. After the gold shell is formed, Au@MNPs show a new absorption peak at ∼540 nm, which is not observed in the nanoparticles before Au coating. From the SEM results, it can be seen that the rough gold shell may cause coupling of adjacent Au nanoparticles and red shift of the absorption peak.

[0063] DNA duplex formation: Single-stranded DNA-SH and DNA-COOH were annealed using DNA annealing buffer (5×) to generate double-stranded DNA (SH-DNA-COOH). The two single-stranded DNA oligonucleotides were diluted in annealing buffer to the same molar concentration (5 μM). The reaction conditions were as follows: denaturation at 95°C for 2 minutes, followed by a temperature decrease of 1°C every 90 seconds, and then a slow decrease to 25°C (the entire cooling process lasted at least 45 minutes). The resulting double-stranded DNA (SH-DNA-COOH) was briefly stored at 4°C for subsequent experiments.

[0064] The DNA sequences used in this example are shown in Table 1 below.

[0065] Table 1

[0066] Activate the sulfhydryl groups at one end of the double-stranded DNA using tris(2-carboxyethyl)phosphine (TCEP) at a final concentration of 10 mM. Incubate at room temperature in the dark for 20 minutes. Purify the activated double-stranded DNA using a CENTRI SPIN-10 column to remove excess TCEP. Perform the purification process at least three times.

[0067] Immunosensing sample protein capture: Au@MNPs and primary antibodies were conjugated using the prepared SH-DNA-COOH duplex. One end of the nucleic acid duplex was linked to the Au@MNPs using an activated thiol group. The DNA was incubated at room temperature for 4 hours, magnetically separated, and washed repeatedly with deionized water to remove unbound nucleic acid duplexes. The carboxyl group at the other end was activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at a final concentration of 10 mM for 30 minutes. The DNA was magnetically separated and washed repeatedly with MEST (0.1 M, pH 5.0) buffer to remove excess EDC and NHS. The DNA was then linked to the amine groups of the primary antibody and incubated at room temperature for 2 hours. The primary antibody-conjugated Au@MNPs complex was then magnetically separated and washed. The double-stranded linker DNA used here can be replaced with materials with controllable photochemical responses to link the gold-coated magnetic nanoparticles and the primary antibody. The primary antibody-conjugated Au@MNPs complex was blocked with magnetic bead blocking buffer for 45 minutes. Subsequently, α-synuclein samples with different concentration gradients were added and incubated at room temperature for 1 hour. Unbound α-synuclein was removed by magnetic separation and PBST washing. Secondary antibodies labeled with signal DNA were added to connect to the captured α-synuclein and incubated at room temperature for 20 minutes. Unbound secondary antibodies were removed by magnetic separation and 1× PBS buffer (pH 7.2) washing. The diversity of the sequence of the signal DNA modified on the secondary antibody provides more possibilities for the joint detection of multiple proteins. Since magnetic nanoparticles will produce irreversible aggregation under the action of centrifugal force, we use enzyme cleavage to remove magnetic nanoparticles. The above-mentioned bound Au@MNPs immune complex was resuspended in the EcoRⅠ enzyme cleavage reaction system: 10×EcoRⅠBuffer (5μL), EcoRⅠ10 5 The enzyme digestion reaction was performed in a water bath with 1 μL of 44 μL of H₂O at 37°C for 1 h. The Au@MNPs were removed by magnetic separation to obtain the supernatant containing the free immune complexes. The free immune complexes were temporarily stored at 4°C for subsequent experiments.

[0068] ImmunoPCR: The resulting immune complex (5 μL) was added to the PCR reaction system: 2×SYBR Green qPCR Master Mix (10 μL), 10 μM Primer 1 (0.4 μL), 10 μM Primer 2 (10 μM) (0.4 μL), immune complex (5 μL), and H2O (4.2 μL), and mixed thoroughly. Subsequently, 10 μL of the reaction mixture was loaded into a droplet generator and centrifuged at 6000 g for 5 minutes to generate droplets. The droplets at the bottom of the centrifuge tube were transferred to a PCR instrument (Bio-Rad CFX96) for amplification. The amplification program was as follows: 25°C for 10 min; 94°C for 30 s; 94°C for 5 s, 60°C for 45 s, and 40×. The oil phase used in the present invention is 7% Abil EM 180 + isopropyl palmitate. At high temperatures, the water-in-oil droplets can remain stable.

[0069] Fluorescent droplet counting: Droplets were transferred to a glass plate and observed using an IX73 inverted fluorescence microscope equipped with excitation filters (BP460-495) and emission filters (BA510-550). All droplets and fluorescent droplets were counted using ImageJ. A standard curve and quantification line were constructed using the concentration of α-synuclein in buffer or serum as the horizontal axis and the corresponding percentage of fluorescent droplets as the vertical axis. H2O was used as a blank control. Each assay was performed at least three times.

[0070] Immunosensing α-Syn Detection: As shown in Figure 3, counting the amplified fluorescent droplets revealed a linear correlation between the α-synuclein concentration in the buffer and the ratio of fluorescent droplets over a dynamic range of 0.1 to 1000 fM, with a linear correlation coefficient >0.99. The limit of detection (LOD) was 43.36 aM, and the limit of quantification (LOQ) was 99.59 aM. As shown in Figure 4, the performance of droplet digital immunosensing in serum samples was comparable to that in buffer. A linear relationship was observed for α-synuclein concentrations between 0.5 and 1000 fM, with a linear correlation coefficient of 0.9904. The limit of detection (LOD) in serum was 173.02 aM, and the limit of quantification (LOQ) was 660.91 aM.

[0071] Example 2

[0072] Specific detection of the ultrasensitive digital immunosensor method of the present invention.

[0073] The present invention evaluated the specificity of droplet digital immunosensing using α-synuclein isoforms β-synuclein and γ-synuclein, as well as other blood proteins: interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), human leukocyte antigen (HLA-DR), and human lipopolysaccharide protein (LBP). All steps were consistent with those in Example 1. As shown in Figure 5, droplet digital immunosensing demonstrated high specificity for α-synuclein.

[0074] Example 3

[0075] The present invention proposes a centrifugal digital immunosensor technology to detect phosphorylated α-synuclein Ser129.

[0076] The present invention detects the phosphorylated Ser129 site of α-synuclein using a recombinant anti-α-synuclein (phospho-S129) antibody as the primary antibody for droplet digital immunosensing. All steps are essentially the same as those in Example 1 above. As shown in Figure 6, the limit of detection (LOD) for phosphorylated α-synuclein Ser129 is 147.23 aM, and the limit of quantification (LOQ) is 1.567 fM.

[0077] The embodiments described above may be further combined or replaced, and the embodiments are merely descriptions of preferred embodiments of the present invention and do not limit the concept and scope of the present invention. Various changes and improvements made to the technical solutions of the present invention by persons of ordinary skill in the art without departing from the design concept of the present invention are within the scope of protection of the present invention. The scope of protection of the present invention is defined by the appended claims and any equivalent technical solutions.

Claims

1. An ultrasensitive digital immunosensing method, characterized in that: The magnetic particles are coupled to the primary antibody; The secondary antibody was modified with single-stranded signal DNA; The primary antibody and the secondary antibody respectively recognize the target protein to form a magnetic particle immune complex with a sandwich structure; The magnetic particles are separated and removed to obtain free immune complexes with sandwich structures; The free sandwich-structured immune complexes are loaded into water-in-oil (W / O) droplets by a droplet generator; Amplify single-stranded signal DNA to generate fluorescent droplets; count the fluorescent droplets to obtain the content of the target protein.

2. The method according to claim 1, characterized in that: The magnetic particles are gold-coated magnetic nanoparticles (Au@MNPs).

3. The method according to claim 1, characterized in that: The magnetic particles and primary antibodies are coupled using double-stranded DNA with different groups at both ends, a gold-sulfur covalent bond is formed between the magnetic particles and the double-stranded DNA, and an amide bond is formed between the primary antibody and the double-stranded DNA.

4. The method according to claim 1, characterized in that: The single-stranded signal DNA modified secondary antibody uses the mutual covalent binding between biotin and streptavidin.

5. The method according to claim 3, characterized in that: The double-stranded linked DNA is treated by a restriction endonuclease-specific digestion method to release the sandwich-structured immune complex.

6. The method according to claim 1, characterized in that: The free immune complexes are loaded into water-in-oil (W / O) droplets by a droplet generator, and the types of droplet generators include centrifugal, microfluidic chip or oscillating.

7. The method according to claim 1, characterized in that: The single-stranded signal DNA amplification includes real-time fluorescence quantitative PCR and loop-mediated isothermal amplification; the DNA amplification dye includes double-stranded DNA binding dye or molecular probe.

8. The method according to claim 1, characterized in that: The target protein may be derived from animals, plants, microorganisms, or artificial products.

9. The method according to claim 1, characterized in that: The target protein is sourced from untreated or pretreated human body fluids, including serum, urine, cerebrospinal fluid, or saliva.

10. The method according to claim 1, characterized in that: Here are the steps: 1) The primary antibody is coupled to gold-coated magnetic nanoparticles using double-stranded linked DNA to capture the target protein; 2) The secondary antibody is modified with single-stranded signal DNA to recognize the target protein and form an immune complex with a gold-coated magnetic nanoparticle-sandwich structure; 3) Use restriction endonucleases to specifically cut the double-stranded DNA to release the sandwich-structured immune complex; 4) using a droplet generator to load the free immune complexes into droplets, so that each droplet contains only one sandwich-structured immune complex; 5) Performing digital PCR amplification on the droplets to generate fluorescent signals; 6) Count the fluorescent droplets and total droplets to obtain the concentration or content of the target protein.

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